The present invention relates to recombinant DNA encoding the BsmAI restriction endonuclease (endonuclease) as well as BsmAI methyltransferase (methylase), as well as expression of BsmAI endonuclease and methylase in E. coli cells containing the recombinant DNA.
BsmAI endonuclease is found in the strain of Bacillus stearothermophilus A664 (New England Biolabs"" strain collection #481). It recognizes the double-stranded DNA sequence 5xe2x80x2 GTCTC 3xe2x80x2 N1/N5 (SEQ ID NO:1) and cleaves downstream sequence at N1 (top strand) and N5 (bottom strand) to generate a 4-base 5xe2x80x2 overhang (/ indicates the cleavage of phosphodiester bond). BsmAI methylase (M.BsmAI) is also found in the same strain.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) on DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2 TTT/AAA 3xe2x80x2 (SEQ ID NO:2), 5xe2x80x2 PuG/GNCCPy 3xe2x80x2 (SEQ ID NO:3) and 5xc2x0 CACNNN/GTG 3xe2x80x2 (SEQ ID NO:4) respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2 G/AATTC 3xe2x80x2 (SEQ ID NO:5).
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78: 1503-1507, (1981)). Since the expressions of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that sometimes positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methylases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (W=A or T) (SEQ ID NO:6) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2 (SEQ ID NO:7)) refractory to NotI digestion (New England Biolabs"" Catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a great commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning BsmAI methylase gene from Bacillus stearothermophilus A664 into E. coli by methylase selection and inverse PCR amplification of the adjacent DNA containing BsmAI restriction endonuclease gene.
At first an ApoI partial genomic DNA library was constructed using the cloning vector PRRS (ApR). No methylase positive clones were identified following the methylase selection method. No resistant clones were found in Aat II, Bg/II, HindIII, KpnI, NdeI, PstI, SacI, SalI, SphI, and XbaI genomic DNA libraries after BsmAI challenge and retransformation with the cloning vector pUC19 (ApR). This negative result indicated that the selection was not strong enough. To increase the selection efficiency, another cloning vector pBR322 with two drug resistance markers was then used for methylase selection. In addition to two drug selection markers, there is a BsmAI site in the rop gene of pBR322. Disruption of the rop gene may further reduce the background in methylase selection.
AatII, ClaI, EcoRI, HindIII, and NdeI genomic DNA libraries were constructed using pBR322 as the cloning vector. Both ApR and TcR markers were used for selection following BsmAI digestion. BsmAI-resistant clones carrying BsmAI methylase gene was discovered in the NdeI DNA library. DNA sequence analysis of the insert and further inverse PCR revealed that BsmAI methylase is comprised of two fused methylases, one of them is a C5 methylase and the other is an amino-methyltransferase.
Since restriction genes are usually located in close proximity to methylase genes, inverse PCR was employed to clone the adjacent DNA surrounding the bsmAIM gene. Open reading frames (ORF) were identified on both sides of the bsmAIM gene. The downstream ORF showed strong homology to a RNA methylase in GenBank and thus it was not the BsmAI endonuclease gene. The upstream ORF did not show significant homology to any gene in Genbank and was predicted to encode the BsmAI endonuclease gene. This ORF was cloned into the expression vector pACYC-T7ter, which was derived from pACYC184, pET11, and pAII17. In order to express bsmAIR gene in E. coli, the bsmAIM gene (M1::M2 fusion) was first cloned in pBR322 to premodify T7 expression host ER2566. BsmAI endonuclease activity was detected in IPTG-induced cell extracts. Three clones with high BsmAI activity were sequenced and confirmed to contain the wild type sequence.